Glucosamine-induced glycation of hydrolysed meat proteins in the presence or absence of transglutaminase: Chemical modifications and taste-enhancing activity

Food Chemistry 197 (2016) 1143–1152

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Glucosamine-induced glycation of hydrolysed meat proteins in the presence or absence of transglutaminase: Chemical modifications and taste-enhancing activity Pui Khoon Hong, Maurice Ndagijimana, Mirko Betti ⇑ Department of Agricultural, Food and Nutritional Science (AFNS), Faculty of Agricultural, Life and Environmental Sciences (ALES), University of Alberta, 4-10 Agriculture/Forestry Centre, Edmonton, Alberta T6G 2P5, Canada

a r t i c l e

i n f o

Article history: Received 6 July 2015 Received in revised form 6 November 2015 Accepted 18 November 2015 Available online 27 November 2015 Chemical compounds studied in this article: Glucosone (PubChem CID: 159630) 3-Deoxyglucosone (PubChem CID: 114839) Glyoxal (PubChem CID: 7860) Methylglyoxal (PubChem CID: 880) Diacetyl (PubChem CID: 650) Keywords: Isoelectric solubilisation and precipitation process Maillard reaction Poultry protein isolate Glucosamine Salty Savoury

a b s t r a c t Salt reduction in food is a challenging task. The food processing sector has adopted taste enhancers to replace salt partially. In this study, a flavour enhancer formulation (liquid seasoning) was produced using enzymatically hydrolysed poultry proteins isolate (PPI). The PPI obtained through the isoelectric solubilisation precipitation process (ISP) was hydrolysed with Alcalase and glycated with glucosamine (GlcN) at moderate temperatures (37/50 °C) in the presence or absence of transglutaminase (TGase). The glycated hydrolysates showed reduced fluorescence advanced glycated end-products (AGE) and a reduced amount of alpha-dicarbonyl compounds (a-DC). An untrained consumer panel ranked the meat protein hydrolysate seasoning saltier than the salty standard seasoning solution (p < 0.05) regardless of GlcN glycation (both tested at 0.3 M Na+). GlcN treatments showed a tendency (p = 0.0593) to increase savouriness. Free glutamic acid and free aspartic acid found in the PPI hydrolysate likely increased the salty perception. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction The global trend to reduce sodium in food is due to health concerns of hypertension and cardiovascular disease. One of the strategies to reduce salt intake is through the addition of flavour enhancers in processed foods. Unlike food flavours, flavour enhancers do not possess flavour or taste themselves, but rather intensify the flavours of other compounds. Common commercial flavour enhancers are inosinates, gluanylates and glutamates. Since consumers are demanding ‘‘natural” food ingredients, several studies explored alternatives such as the deamidation of wheat gluten protein (Liao et al., 2010; Schlichtherle-Cerny & Amado, 2002), enzymatic hydrolysis of shrimp protein (Cheung & Li-Chan, 2014), and chicken muscle protein (Maehashi, Matsuzaki, Yamamoto, & ⇑ Corresponding author. E-mail address: [email protected] (M. Betti). http://dx.doi.org/10.1016/j.foodchem.2015.11.096 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

Udaka, 1999). These peptides and proteins were reported to enhance the umami (savoury) and salty taste. For instance, Maehashi et al. (1999) isolated an umami fraction from chicken proteins hydrolysed with bromelain; within this fraction several di- and tri-peptides containing glutamic acid (i.e. Glu–Glu and Ala-Glu-Asp, respectively) were identified. These peptides demonstrated an increase to the umami taste when used in combination with 50 -inosine monophosphate (IMP), a commercial flavour enhancer. Also, meat proteins contain high levels of glutamic and aspartic acid that can be released with chemical or enzymatic hydrolysis to increase the umami taste. The recent interest in the valorisation of meat and fish processing by-products has led to the production of muscle protein isolates recovered through the isoelectric solubilisation and precipitation process (ISP). This can be a suitable and an economic protein-based biomass to hydrolyse and unleash the potential of glutamic acid in amplifying the deliciousness of food. For instance, Hrynets, Omana, Xu, and Betti

1144

P.K. Hong et al. / Food Chemistry 197 (2016) 1143–1152

(2011) found that glutamic acid significantly increased after the ISP of mechanically separated poultry meat (MSPM) compared to the starting material. Even without hydrolysis, poultry protein isolate (PPI) was successfully incorporated in chicken patties with positive consumer sensory acceptability (Khiari, Pietrasik, Gaudette, & Betti, 2014). The Maillard reaction, also known as glycation, is a common process to generate food flavours. This reaction involves the condensation of the carbonyl group of a reducing sugar with an amino compound, followed by the degradation of the condensation products to alpha-dicarbonyl (a-DCs). Subsequently, a-DCs react with other compounds such as amines, amino acids, aldehydes, hydrogen sulphide and ammonia, leading to many important classes of flavour compounds, including furans, pyrazines, pyrroles, oxazoles, thiophenes, thiazoles and other heterocyclic compounds. Several studies have been conducted in a simple model system consisting of an amino acid/peptide and a reducing sugar to generate specific flavour compounds (Lee, Jo, & Kim, 2010; Xu et al., 2013). Recently, some compounds generated through the Maillard reaction, specifically the so called ‘‘Maillard reacted peptides” (MRP), possess flavour-enhancing properties. For instance, soy protein isolate glycated with various reducing sugars (Katsumata et al., 2008; Lan et al., 2010; Ogasawara, Katsumata, & Egi, 2006; Song et al., 2013) as well as xylose conjugated with sunflower protein hydrolysate (Eric et al., 2013) yielded a mixture of MRP (modified peptides + glycopeptides), which not only enhanced the savoury taste, but also increased the intensity of the mouthfulness and continuity sensations known as ‘‘kokumi”. On the other hand, despite the usefulness of the Maillard reaction to generate both flavours and MRP, one of the negative effects is the production of browning compounds. These compounds are usually associated with toxic substances, such as acrylamide and advanced glycation endproducts (AGEs). This becomes particularly relevant when the Maillard reaction is conducted at elevated temperatures (i.e. >100 °C), especially during cooking (Mottram, Wedzicha, & Dodson, 2002; Poulsen et al., 2013; Stadler et al., 2002). Consumers demand the production of ‘‘clean” and ‘‘natural” label ingredients. Therefore, alternative approaches which minimise the formation of browning compounds whilst increasing the production of the flavour enhancing MRP are of interest. In this aspect, Hong, Gottardi, Ndagijimana, and Betti (2014) and Hrynets, Ndagijimana, and Betti (2013) had successfully glycated fish gelatin hydrolysate and myofibrillar proteins at moderate temperature (37 °C) to generate modified peptides and proteins with enhanced bioactivity (i.e. antioxidant capacity) and functionality (i.e. solubility), respectively. This was likely due to the use of the highly reactive amino sugar glucosamine (GlcN) in concert with the enzyme transglutaminase (TGase). Here, novel glycoconjugates may be produced due to the ability of TGase to catalyse the transfer of an acyl group from a glutamine amino acid in a protein or peptide sequence to the amino group of GlcN, forming a stable isopeptide bond. The effectiveness of using GlcN as a way to modify peptides has been already exploited by Katsumata et al. (2008). In this study a fraction of soy protein hydrolysate was glycated at 95 °C for 4.5 h generating MRPs that modulate the salty taste. However, at 95 °C, browning and heterocyclic compounds may be generated. It is our intention in this current research to generate MRP using GlcN at significantly lower temperatures that still amplify saltiness and increase savouriness. Our main objective was to evaluate the chemical and enzymatic modifications of hydrolysed poultry meat proteins in response to GlcN glycation in the presence or absence of TGase at moderate temperatures (37, 50 °C). We examined how this affected the saltiness and savoury perception of the modified hydrolysates in the seasoning composition. The PPI extracted at a pilot plant facility with the ISP process was hydrolysed with a commercial protease

Alcalase and subjected to GlcN glycation treatment in the presence or absence of TGase. Then, it was used to formulate a liquid seasoning composition which was subsequently used for a consumer sensory evaluation. Chemical changes regarding composition and structure due to GlcN treatments were analysed and correlated to the consumer panel results. 2. Materials and methods 2.1. Chemicals Mechanically separated turkey meat (MSTM) was obtained from Lilydale Inc. (Edmonton, AB, Canada). Food grade Alcalase was a gift from Novozymes (Franklinton, NC). GlcN hydrochloride and reduced glutathione (GSH) were purchased from PureBulk (Roseburg, OR). Food grade microbial TGase (ACTIVAÒ TI) was manufactured by Ajinomoto (France). Food grade hydrochloric acid, sodium hydroxide and acetic acid were purchased from Fisher Scientific (Fisher Scientific Company, Ottawa, ON). Monosodium glutamate was purchased from Ajinomoto (La Victoria, Peru). Chemicals used for poultry protein isolation and sensory evaluation were of food grade. All other chemicals and solvents used in other analyses and liquid chromatography were of analytical grade or HPLC grade. They were purchased from Sigma–Aldrich (Sigma– Aldrich, St. Louis, MO) and Fisher Scientific (Ottawa, ON, Canada). 2.2. Experimental design Poultry protein was isolated from commercial MSTM using the acid-aided ISP process. The poultry protein was extracted from 80 kg of MSTM in four batches. Subsequently, they were pooled into one batch, stored at 20 °C and used within 3 months. Extracted protein (6.0–6.5% w/v) was hydrolysed with Alcalase in 11 batches and then pooled. Following enzymatic hydrolysis, the glycation treatment was performed. The hydrolysate (5% protein) was incubated with GlcN either in the presence or absence of TGase at moderate temperatures (37 or 50 °C) and at pH 7.0 ± 0.5, resulting in the following treatments: native hydrolysate 37 °C, native hydrolysate 50 °C, glycated hydrolysate 37 °C in the absence of TGase, glycated hydrolysate 37 °C in the presence of TGase, and glycated hydrolysate 50 °C in the absence of TGase. For the purpose of chemical analysis, all treatments were acidified to pH 4.9 with food grade HCl at the end point of incubation, pasteurised at 80 °C for 10 min, lyophilised and kept at 18 °C until used. Chemical modification of the hydrolysate in response to GlcN glycation was assessed using spectroscopic techniques (see Section 2.4) and a-dicarbonyl formation (Section 2.5). On the other hand, for sensory evaluation, the native and glycated hydrolysates were used to formulate a composition (a liquid seasoning) similar to the ones commercially available (i.e. Maggi seasoning). Therefore, acetic acid and HCl (food grade acids) were used to adjust the pH of the seasonings to 4.9 at the end point of incubation and before they were pasteurised (80 °C for 10 min). These liquid seasonings were kept frozen at 18 °C and used within two weeks. Protein, pH and Na+ content were standardised to 3% w/v, 4.9 and 0.3 M, respectively, prior to sensory evaluation. The taste enhancing properties (saltiness and savouriness) were studied using ranking tests. Seasoning control samples containing glutamate (‘‘savoury seasoning”), glutathione (‘‘kokumi seasoning”), and, a mixture of the two (‘‘kokumi + savoury seasoning”) were also produced and standardised at the same levels of Na+ and pH. For the evaluation of saltiness and savouriness of the seasonings, a randomised complete design was used with sample treatment as experimental unit. Participants were asked to evaluate the intensity of saltiness and savoury tastes perceived in two separate sessions.

P.K. Hong et al. / Food Chemistry 197 (2016) 1143–1152

2.2.1. Poultry protein isolate Poultry protein was obtained from the MSTM (moisture 67.0%, protein 14.1%, fat 17.6%, ash 1.2%, carbohydrate 0.1%) using the ISP procedures. The PPI was prepared at a certified food-grade pilot plant at the Food Processing Development Centre (FPDC) in Leduc, AB, Canada as according to the optimised method of Khiari et al. (2014), Fig. S-1 with modifications. The extraction was carried out in 4 batches. For each batch, about 20 kg of minced MSTM were mixed with ice-water mixture at a 1:5 weight ratio. Citric acid (0.038% w/w) was added into the mixture prior to homogenisation to help in reducing the total lipid and pigment in MSTM. Fat was scooped out from the mixture 30 min after resting from homogenisation at 4 °C. The protein solution was adjusted to pH 2.5 with 2 M HCl. The soluble proteins were obtained by centrifugation (bowl speed 8500 rpm; Westfalia Separator AG, Model NA 7-06-076; Oelde, Germany) to remove the insoluble particles. Then, the soluble proteins were precipitated at pH 5.2 (isoelectric point) with 2 M NaOH, followed by centrifugation. The precipitate was collected and added with water (0–4 °C) at a ratio of 1:1 (w/w) at pH 5.2. The mixture was centrifuged for the second time in order to remove soluble pigments and to collect the precipitated protein. Cryoprotectants (0.3% w/w sodium tripolyphosphate, 0.03% w/w sodium nitrite and 0.4% w/w sodium bicarbonate) were added to the PPI (w/w: moisture 91.4%, protein 7%), they were pooled and stored at 18 °C and used within 3 months. 2.2.2. Hydrolysis of PPI The preparation of the PPI hydrolysates was carried out in 11 batches at a designated food preparation laboratory at the University of Alberta. The food grade PPI hydrolysate was prepared by hydrolysing with Alcalase (EC 3.4.21.62, 15 U/g PPI hydrolysate protein) at 50 °C for 3.5 h (pH 7.5–8.0) with constant stirring. In a preliminary study, the PPI had a poor solubility problem, probably due to freezing denaturation during storage without the addition of sucrose as a cryoprotectant component. Therefore, prior to hydrolysis, the PPI solution was adjusted to approximately pH 10.5 for 30 min (Fig. S-1b) to encourage protein unfolding to improve its solubility; then it was adjusted to pH 7.5–8.0 and 6.0–6.5% w/v protein for hydrolysis. The pH of the hydrolysate was maintained at 7.5–8.0 by adding 2 M sodium hydroxide. The hydrolysate was heated to 80 °C for 10 min and cooled down to 4 °C. Then, it was left at 4 °C overnight for phase separation. The clear supernatant was collected and filtered with filter paper to remove impurities. The filtrate (w/v: protein 6.3%, Na+ 0.4 M) was pooled and stored at 18 °C until use. The pH stat method was used to assess the degree of hydrolysis (% DH) of three random batches of PPI hydrolysates. The DH was calculated as according to Adler-Nissen (1986). 2.2.3. Crude protein and Na+ determination Crude protein was measured using a nitrogen analyser (Leco TruSpec C/N analyzer; Leco Corporation, St. Joseph, MI) and applying a conversion factor of 6.25 on the percentage of N. During the ISP process NaCl is produced. Hence, monitoring the Na+ content is important to formulate a standardised seasoning composition for the sensory evaluation. Na+ content was determined with atomic absorption spectroscopy (Varian AA240FS sequential atomic absorption spectrometer; Agilent Technologies, Santa Clara, CA) at 589.6 nm using a fuel lean air/acetylene flame according to the manufacturer’s manual. 2.2.4. Preparation of glycoconjugated hydrolysates The preparation of glycoconjugated hydrolysates was carried out in 3 batches for each treatment at a designated food preparation laboratory at the University of Alberta. For each batch, about 0.5 L of the PPI hydrolysate solution containing 5% w/v protein

1145

were added to GlcN at a weight ratio of 4:1. Sodium hydroxide (2 M) was used to adjust the pH to 7.0 ± 0.5 (Fig. S-1c). Hydrolysates were conjugated with GlcN by glycation without TGase, at 37 °C and 50 °C, whilst another glycated hydrolysate treatment was incubated at 37 °C in the presence of TGase. In this case, food-grade microbial TGase (26 ± 5 unit/g enzyme; at a ratio of 2 unit/g protein) was used. As for controls, the PPI hydrolysates were incubated in the same manner as indicated above without GlcN. GlcN samples were incubated at 37 °C and 50 °C as controls for the glycated hydrolysates. All treatments were incubated for 3.5 h, then, they were adjusted to pH 4.9 with 2 M HCl and pasteurised at 80 °C for 10 min. Subsequently, they were cooled to 4 °C and filtered with a Minimate TFF Capsule fitted with an OmegaTM MWCO 10 kDa membrane (Pall Canada Ltd., Quebec, QC, Canada). All the treatments were pooled and lyophilised and stored at 18 °C until used for chemical analysis. 2.3. Molecular weight characterisation of PPI hydrolysate The molecular weight distribution of the samples was analysed by matrix-assisted laser desorption ionisation time-offlight mass spectrometry (MALDI–TOF-MS) as described by Hong et al. (2014). Freeze-dried PPI hydrolysate was diluted in acetonitrile:water (1:1) and acidified with 0.1% trifluoroacetic acid. One microlitre of the diluted hydrolysate was mixed with 1 lL of a-cyano-4-hydroxycinnamic acid (10 mg/mL 4-HCCA in acetonitrile:water (1:1) acidified with 0.1% trifluoroacetic acid). One microlitre of the mixture was spotted onto a stainless steel target plate and allowed to air dry. The mass spectra were acquired with a Bruker Ultraflex MALDI–TOF/TOF (Bruker Daltonic, GmbH, Rheinstetten, Germany) in positive ion mode (25 kV). An external calibration was carried out using a standard peptide mixture. 2.4. Spectral characteristics of hydrolysed poultry protein in relation to GlcN treatment 2.4.1. UV–vis profile In order to monitor the progress of Maillard reaction in the GlcN modified hydrolysates, all the treatments were scanned at UV–visible wavelengths. Approximately 5 mg/mL of each glycated samples (1.25 mg/mL for GlcN samples) in a 1-cm quartz cuvette was scanned at k = 200–500 nm using the Spectramax M3 multimode microplate reader (Molecular Devices, Sunnyvale, CA). The intensity of each of the samples was recorded and compared with other treatments. 2.4.2. AGE-fluorescence compounds The glycoconjugated hydrolysates were diluted to 5 mg/mL, whereas the GlcN samples were diluted to 1.25 mg/mL which correspond to the ratio of protein:GlcN = 4:1 used during glycation of hydrolysates. The fluorescence emission spectra (excitation = 347 nm; emission = 400–600 nm) were assessed using a 1cm quartz cuvette and the Spectramax M3 multi-mode microplate reader (Molecular Devices, Sunnyvale, CA). 2.5. Identification of a-DC 2.5.1. Extraction of a-DC The extraction of the a-DC from glycated hydrolysates was based on the 3-step procedure described by da Silva Ferreira, Reis, Rodrigues, Oliveira, and de Pinho (2007) and modified by Papetti, Mascherpa, Marrubini, and Gazzani (2013). Exactly 6 mL of lyophilised GlcN modified hydrolysates (20 mg/mL, dissolved in DI water) from each treatments were passed through a preconditioned Sep-Pak C-18 cartridge (flow rate = 2 mL/min, 10 mL

1146

P.K. Hong et al. / Food Chemistry 197 (2016) 1143–1152

methanol and 20 mL DI water). Each of the polar fractions obtained was spiked with 6 mg of 1,2-diaminobenzene (ophenylenediamine (OPD)) to derivatise the a-DC into their respective quinoxaline derivatives at 37 °C for 1 h (pH 3.00 ± 0.02). Later, the derivatised samples were passed through another C-18 cartridge with 4 mL of 90:10 v/v pure methanol/water mixture (flow rate 2 mL/min). The first 1 mL was discarded and the subsequent 2 mL eluted from the C-18 cartridge were collected. Separation of the a-DC was carried out using an Ascentis Express Peptide ESC18 column (150 mm  4.6 mm ID, 2.7 lm; Sigma–Aldrich, St. Louis, MO) connected to an ultra-high-performance liquid chromatography (UHPLC) apparatus (Shimadzu, Columbia, MD). The UHPLC system comprised a binary pump, a degasser, an autosampler, an oven set at 25.0 ± 0.5 °C, and a diode array detector set at 314 nm. The separation procedures were carried out as according to Papetti et al. (2013). All the samples were injected in triplicate. The identification of the a-DCs was based on the retention times in comparison to the pure standards (quinoxaline derivatives). For the confirmation of the identified a-DC derivatives, peaks were collected from the UHPLC and analysed by Orbitrap mass spectrometry (Section 2.5.2). The quantification of the a-DCs (as quinoxaline derivatives) was achieved by external calibration. Quinoxaline derivatives of glucosone (11.1–89.1 mg/L), 3-deoxyglucosone (20.3–648.6 mg/L), glyoxal (0.1–11.6 mg/L), methylglyoxal (0.1–3.6 mg/L), and diacetyl (0.4–1.7 mg/L) were prepared. The limit of detection (LOD) determined from the triplicate blanks was as follows: 675 (glucosone), 375 (3-deoxyglucosone), 34 (glyoxal), 1 (methylglyoxal) and 6 ng/L (diacetyl); whereas their limit of quantification (LOQ) was 2046 (glucosone), 1137 (3-deoxyglucosone), 104 (glyoxal), 4 (methylglyoxal), and 19 ng/L (diacetyl). 2.5.2. Direct infusion Orbitrap mass spectrometry analyses (DIMS) Fractions obtained from UHPLC were diluted to a volume ratio of 1:1 (methanol:water). DIMS was performed as described by Hrynets, Ndagijimana, and Betti (2015a) on a LTQ Orbitrap XL (Thermo Scientific, San Jose, CA,) operated in electrospray ionisation (ESI) mode. Data were acquired and processed using Xcalibur software (Thermo Scientific). The retention times, precursor ions and fragmentation patterns (tandem MS) of the a-DC quinoxaline derivatives are listed in Table S-1.

about 2.25 L of the PPI hydrolysate (containing 5% protein) were glycated with GlcN; as for GlcN control, only the GlcN solution incubated at 50 °C was prepared for sensory evaluation. Acetic acid (2 M) was added (1% v/v) and 2 M HCl were added to adjust to pH 4.9 towards the end of incubation. Acetic acid was added in order to benchmark a commercial liquid seasoning (Maggi Seasoning) in which acetic acid is one of the main ingredients. The hydrolysates and controls were pasteurised at 80 °C for 10 min, cooled to 4 °C and stored at 18 °C. Samples were pooled according to treatment and consumed within two weeks. The hydrolysate and glycoconjugated hydrolysates were diluted to a final protein content of approximately 3.0% w/v prior to sensory evaluation. On the other hand, the sodium content in all the treatments was standardised to approximately 0.3 M Na+. Four standard liquid seasonings were prepared as controls (Table 1). They consisted of ‘sodium seasoning’ (0.3 M Na+), ‘savoury seasoning’ (Na+ + monosodium glutamate (MSG), 3 mM), ‘kokumi seasoning’ (0.3 M Na+ + reduced glutathione (GSH), 5 mM) and ‘savoury + kokumi seasoning’ (0.3 M Na+ + 3 mM MSG + 5 mM GSH). A total of 125 consumers (age 18–69, Table S-2) consisting of students and staff from the University of Alberta were hired to participate in two sensory evaluation sessions. They met the following criteria based on a questionnaire given to the consumers: healthy, free from food allergy, sensitivities, intolerance or discomfort to the ingredients used in the sensory evaluation. The sensory evaluation study was approved by the University of Alberta’s Human Ethics Research Office and written consent was obtained from participants during recruitment. Samples were served warm (50–60 °C) in covered plastic cups (approximately 15 mL in each cup) labelled with randomised three digits. Participants were requested to evaluate the samples using the sit-and-spit method; they were told to hold each sample in their mouth for approximately 10 s before spitting it out. All sensory evaluation sessions were carried out in red-lit booths. In the first session, 64 consumers were asked to rank the saltiness intensity of the 10 samples served randomly; another 61 consumers were asked to rank the savoury intensity in the second session. Score 1 was given to samples with the highest intensity whereas score 10 was given to samples possessing the lowest intensity tested.

2.6. Quantification of savoury free amino acids in response to GlcN treatments The free amino acid content of the GlcN modified hydrolysates, native hydrolysates and GlcN were performed as described by Sedgwick, Fenton, and Thompson (1991) with pre-column ophthaldialdehyde (OPA) derivatisation. Calibration was done by using 0.1 lM b-amino-n-butyric acid (BABA) as internal standard, and amino acid standard mixture (Sigma–Aldrich, St. Louis, MO) supplemented with glutamine, asparagine, and tryptophan. Data were acquired and processed using Galaxie chromatography software (Agilent Technologies, Santa Clara, CA). The total umami amino acids was the sum of free glutamic acid and free aspartic acid.

Table 1 Seasoning composition used for sensory evaluation. Treatments

Native hydrolysate 37 °C Native hydrolysate 50 °C Glycated hydrolysate 37 °C in absence of TGase Glycated hydrolysate 37 °C in presence of TGase Glycated hydrolysate 50 °C in absence of TGase Glucosamine 50 °C Salty seasoning (sodium) Savoury seasoning (sodium + monosodium glutamate (MSG)) Kokumi seasoning (sodium + glutathione (GSH)) Savoury + kokumi seasoning (sodium + MSG + GSH)

2.7. Seasoning preparation and sensory evaluation on the saltiness and savouriness intensity In order to assess the potential of the hydrolysed PPI as a taste enhancer, the native hydrolysates and the glycated hydrolysates were formulated similarly to the seasoning liquids available on the market. The glycated hydrolysates were prepared as mentioned in Section 2.2.4 with slight modification prior to pasteurisation. Two batches per treatment were prepared, for each batch,

* ** ***

0.7% Na+ w/v = 0.3 M Na+. 0.05% MSG w/v = 3 mM MSG. 0.15% GSH w/v = 5 mM GSH.

Concentration (% w/v) Na+*

Protein from hydrolysate

Taste enhancer

0.7 0.7 0.7

3 3 3

– – –

0.7

3

–

0.7

3

–

0.7 0.7 0.7

– – –

– – 0.05 MSG**

0.7

–

0.15 GSH***

0.7

–

0.05 MSG + 0.15 GSH

1147

P.K. Hong et al. / Food Chemistry 197 (2016) 1143–1152

was also used to hydrolyse the PPI (data not shown). In a preliminary sensory panel, this enzyme has an intrinsic savoury taste that could have invalidated our study (data not shown); therefore it was not used in the current study. Based on the optimised hydrolysis duration (Fig. 1a), the 3.5 h time point was chosen as it represented the hydrolysis plateau, with an average degree of hydrolysis (DH) of 6.9 ± 1.0%. The DH obtained in this study was not particularly high, likely due to the combined effect of ISP and freezedenaturation that occurred during freezing storage, resulting in protein aggregation with subsequent negative effects on the hydrolysis process. Several research teams have targeted peptides at the MW range of 1000–5000 Da as a prerequisite in the production of kokumi taste-enhancing peptides (Eric et al., 2013; Katsumata et al., 2008; Lan et al., 2010; Ogasawara et al., 2006; Song et al., 2013). The m/z of the PPI hydrolysate was 448.264– 2905.925 (Fig. 1b), which was confined to the range mentioned.

2.8. Data statistical analysis The means of a-DC and free amino acid content were analysed with one-way ANOVA and separated by Duncan test. The means were considered statistically significant at p 6 0.05. The data obtained from the sensory evaluation sessions were compiled in the form of a box plot. Then, the mean rank scores obtained from sensory evaluation sessions were analysed with Friedman’s test and separated with Bonferroni adjustment. The mean rank scores were considered statistically significant at p 6 0.05. 3. Results and discussions 3.1. Characterisation of PPI hydrolysate The PPI was hydrolysed with the endopeptidase Alcalase, an alkaline bacterial protease produced from Bacillus licheniformis. This enzyme was selected based on its ability to release savoury amino acids; for instance it has been successfully used in wheat gluten hydrolysis to release glutamic acid, producing a potential taste enhancing hydrolysate (Koo et al., 2014). Flavourzyme, a fungal protease/peptidase complex produced from Aspergillus oryzae,

3.2. UV–VIS and fluorescence profiles of hydrolysed poultry proteins in response to GlcN treatments UV–visible analysis is a simple yet rapid method to monitor and characterise the changes occurring during the Maillard reaction.

(a) 8

6

4

2 3.5 h

0 0

5

10

15

20

Hydrolysis duration, h

671.929 682.989 695.716

x10 4

2905.925

0.4

1669.586 1723.687

0.6

551.918

0.8

1185.888 1226.154 1265.135 1274.855 1326.849 1355.796 1405.020 1449.617 1471.937 1512.572 1568.186 1592.380

593.107

1.0

1030.381 1086.817

950.039

1.2

748.945 793.249 816.251 866.064846.650 913.725

Intens. [a.u.]

(b)

0.2

0.0 500

1000

1500

2000

2500

3000

3500

4000

4500

m/z

Fig. 1. (a) Hydrolysis curve of poultry protein isolate and Alcalase for 17 h (15 U/g protein, pH 7.5–8.0, 50 °C). Means ± standard deviation of three random batches of PPI hydrolysed with Alcalase were plotted. (b) MALDI–TOF-MS spectrum of poultry protein hydrolysed with Alcalase.

1148

P.K. Hong et al. / Food Chemistry 197 (2016) 1143–1152

(a) 4

Absorbance

Native hydrolysate 37 ˚C Native hydrolysate 50 ˚C

3

Glycated hydrolysate 37 ˚C in absence of TGase Glycated hydrolysate 37 ˚C in presence of TGase

2

Glycated hydrolysate 50 ˚C in absence of TGase Glucosamine 37 ˚C Glucosamine 50 ˚C

1

0 200

250

300

450

500

Native hydrolysate 37 ˚C

(b)

Native hydrolysate 50 ˚C

1500 Relative Fluorescence Unit

350 400 Wavelength, nm

Glycated hydrolysate 37 ˚C in absence of TGase Glycated hydrolysate 37 ˚C in presence of TGase Glycated hydrolysate 50 ˚C in absence of TGase

1000

Glucosamine 37 ˚C Glucosamine 50 ˚C

500

450 nm region, with a maximum emission at 410–430 nm, whereas the native hydrolysates did not show any emission (Fig. 2b). As expected, glycation of hydrolysed poultry meat proteins conducted at 50 °C resulted in the highest peak emission, indicating a higher production of AGEs compared to the other treatments. Several fluorescent AGEs could be responsible for the increased fluorescence intensity observed in response to GlcN glycation, possibly including pentosidine, argpyrimidine, pirropyridine, pentodilysine, and also the cyclocondensation products from the GlcN auto-reaction as described previously for UV–Vis analysis (Ferrer et al., 2005; Wilker, Chellan, Arnold, & Nagaraj, 2001). When comparing GlcN incubation alone with GlcN incubated with poultry meat protein peptides, the latter produced more AGEs. The reason for this phenomenon is likely due to a higher amount of accessible –NH2 groups in the system containing both peptides and GlcN. In summary, the spectroscopic analyses indicated that hydrolysed poultry proteins are chemically modified in response to GlcN glycation and the extent of modification was greater at 50 °C. Production of AGEs was lower at 37 °C compared to at 50 °C, indicating that a lower temperature would be more favourable to produce MRP from a food safety point of view. 3.3. Identification and quantitation of the major a-DC

0 400

450

500

550

600

Emmission Wavelength, nm

Fig. 2. Spectral characteristics of hydrolysates, glycated hydrolysates and glucosamine solutions. (a) Ultra-violet spectra (200–500 nm), (b) AGE-fluorescence spectra (excitation = 347 nm, emission = 400–600 nm).

Fig. 2a is the UV spectra of native and GlcN-modified hydrolysates in the presence and absence of TGase, and also GlcN samples incubated alone at the same concentration used in the glycation treatment. In all the treatments two peaks were present. The first sharp peak was in the 220–230 nm region, and the second one was approximately in the 270–280 nm region. The changes of absorbance in relation to GlcN glycation were prominent in the second peak, and related to aromatic amino acids like tryptophan and tyrosine as well as fructosazines and other condensation products resulting from GlcN cyclocondensation (Hrynets et al., 2015a). GlcN incubated alone without hydrolysates at 37 and 50 °C showed lower absorbance at 280 nm, whereas native hydrolysates along with the glycated samples at 37 °C showed higher absorbance values. The highest absorbance was observed when the native hydrolysates were glycated with GlcN at 50 °C. Glycation performed at 37 °C in the presence or absence of TGase did not change the spectral profiles compared to the native hydrolysates. This was contrary to when glycation was carried at 50 °C, where the prominent increase in absorbance observed at 280 nm was likely due to the formation cyclocondensation products (pyrazines) from GlcN and GlcN self-reaction, GlcN and a-DCs reaction (Hrynets et al., 2015a) as well as Strecker degradation, a reaction which occurs between a-DCs and free amino acids and peptides. Fluorescence emission is a good indicator of formation of AGEs (Matiacevich & Buera, 2006) as products of the Maillard reaction. AGEs are normally produced when a-DCs, like 3-deoxyglucosone, attack the free amino groups of peptides and proteins. Samples were excited at 347 nm and emitted at 400–600 nm, these were the typical wavelengths used to assess the AGEs of the Maillard reaction (Ferrer, Alegria, Farre, Clemente, & Calvo, 2005; Morales & Jimenez-Perez, 2001). All the glycoconjugated hydrolysates and the GlcN solutions showed a broad emission peak in the 400–

As previously discussed, a-DCs are very important intermediate products from the Maillard reaction that have the ability to condense with free amino groups in proteins and peptides inducing important structural modifications. They are the precursors of browning and flavouring compounds during the Maillard reaction. As reported by Hrynets, Ndagijimana, and Betti (2015a, 2015b), since GlcN is a Heyns compound, it has the ability to produce significant a-DCs at 37 °C that can then condense with free amino groups. These modifications may be crucial to produce MRP (modified peptides and glycopeptides) that possess flavour-enhancing properties. Fig. 3 illustrates the representative UHPLC chromatograms of collected a-DC peaks and a-DCs produced from the treatments. For confirmation of the peaks for which commercial standards were available, the major peaks were collected and subjected to MS analyses. Glucosone, 3-deoxyglucosone, glyoxal, methylglyoxal and diacetyl were identified and quantified in all the glycated treatments (Table 2), whereas no a-DCs were detected in native hydrolysates. The amount of glucosone detected was significantly different among the treatments. Glycated hydrolysates produced significantly greater glucosone content than the GlcN incubated at 37 °C (p < 0.05) but was not statistically different from the GlcN incubated at the higher temperature (50 °C). Glycation in the absence of TGase conducted at 50 °C produced almost three times greater 3-deoxyglucosone (188 ± 37 mg/L) compared to other glycation treatments conducted at 37 °C. Furthermore, the concentration of 3-deoxyglucosone was greater in the treatments containing the hydrolysed meat proteins than the GlcN incubated alone, likely due to the higher free amino groups available for the Maillard reaction. A similar result was obtained by Hrynets et al. (2015b). 3Deoxyglucosone is a predominant a-DC in food such as fruit juice (not detected–410 mg/L), balsamic vinegar (median 361 mg/L), and bread (13–619 mg/kg); it was also detected at 212 mg/L in liquid condiments/seasonings (Degen, Hellwig, & Henle, 2012). In the current study, the amount of 3-deoxyglucosone found was less than in the commercial food products mentioned. The glyoxal, methylglyoxal and diacetyl content in the treatments were considered as low, ranging between 0.2 and 1.2 mg per L. The level of these short chain a-DC found in the current study were lower than the commercial balsamic vinegars (1.3– 27.9 mg/L) (Papetti et al., 2013); however, they were conforming

1149

P.K. Hong et al. / Food Chemistry 197 (2016) 1143–1152

Fig. 3. (a) UHPLC chromatogram of a-dicarbonyl compound standards: 1 = glucosone (21 min), 2 = 3-deoxyglucosone (33 min), 3 = glyoxal (66 min), 4 = methylglyoxal (79 min), and, 5 = diacetyl (87 min). (b) UHPLC chromatogram of a-dicarbonyl compounds in glycated hydrolysates and GlcN solutions (n = 3): 1 = glucosone, 2 = 3deoxyglucosone, 3 = glyoxal, 4 = methylglyoxal, and, 5 = diacetyl.

Table 2 Major a-dicarbonyl compounds identified in the glucosamine solutions and the glycated hydrolysates produced at moderate temperatures (37 °C and 50 °C). Treatments

a-Dicarbonyl compounds (mg/L) Glucosone

Glycated hydrolysate 37 °C in absence of TGase Glycated hydrolysate 37 °C in presence of TGase Glycated hydrolysate 50 °C in absence of TGase Glucosamine 37 °C Glucosamine 50 °C p-Value

b

57 ± 5 66 ± 9ab 78 ± 10a 29 ± 2c 70 ± 7ab <0.001

3-Deoxyglucosone b

67 ± 5 71 ± 10b 188 ± 37a 9 ± 2c 17 ± 3c <0.001

Glyoxal

Methylglyoxal c

0.3 ± 0.1 0.2 ± 0.1c 0.5 ± 0.1b 1.1 ± 0.1a 1.2 ± 0.2a <0.001

bc

0.3 ± 0.0 0.2 ± 0.0c 0.6 ± 0.1a 0.3 ± 0.0b 0.3 ± 0.0bc <0.001

Diacetyl 0.6 ± 0.1b 0.6 ± 0.1b 1.1 ± 0.1a 1.1 ± 0.3a 0.4 ± 0.1b 0.001

Means ± standard deviation; n = 3. Means with different letters in the same column are significantly different (p < 0.05).

to the range reported for coffee brew, barley coffee brew and soy sauce (0.1–2.7 mg/L) (Papetti, Mascherpa, & Gazzani, 2014). Glyoxal and methylglyoxal are described as having mild sour, pungent and slightly nutty aromas (Kokkinidou, 2013). The formation of glyoxal likely originated from the cleavage at the C2–C3 bond of glucosone (Degen et al., 2012; Hofmann, Bors, & Stettmaier, 1999). A significantly higher content of glyoxal was observed in GlcN incubated at 37 °C and 50 °C (Table 2, p < 0.05). On the other hand, the highest 3-deoxyglucosone was found in the glycation treatment at 50 °C, resulting in the greatest methylglyoxal level.

This makes sense, since 3-deoxyglucosone serves as a precursor for the production of methylglyoxal (pyruvaldehyde) (Weenen, 1998; Yaylayan & Keyhani, 2000) through the fragmentation of 3-deoxyglucosone by retroaldol condensation (Thornalley, Langborg, & Minhas, 1999). In summary, these a-DC results agree with the spectroscopic analysis reported in the previous section. It highlighted that the major modifications observed at 50 °C were at least in part due to a higher production of a-DCs. Glycation conducted at 37 °C also produced a-DCs with the potential of inducing structural

1150

P.K. Hong et al. / Food Chemistry 197 (2016) 1143–1152

a

ab

abc

bcd

Native hydrolysate 50 ˚C

Glycated hydrolysate 37 ˚C in absence of TGase

Glycated hydrolysate 37 ˚C in presence of TGase

Glycated hydrolysate 50 ˚C in absence of TGase

Glucosamine 50 ˚C

d

cd

bcd bcd

Kokumi seasoning

abcd

10

Savoury seasoning

abc

Native hydrolysate 37 ˚C

Rank scores for perceived intensity

p= 0.0593

p<0.001

(a) 9 8 7 6 5 4 3 2 1

Saltiness

Savoury+Kokumi seasoning

Kokumi seasoning

Salty seasoning

Savoury seasoning

Glucosamine 50 ˚C

Glycated hydrolysate 50 ˚C in absence of TGase

Glycated hydrolysate 37 ˚C in absence of TGase

Glycated hydrolysate 37 ˚C in presence of TGase

Native hydrolysate 50 ˚C

Native hydrolysate 37 ˚C

Savoury+Kokumi seasoning

Salty seasoning

0

Savoury

(b) 300

Native hydrolysate 37 ˚C

Concentration, mg/L

250

Native hydrolysate 50 ˚C

200

Glycated hydrolysate 37 ˚C in the absence of TGase

150

100

Glycated hydrolysate 50 ˚C in the absence of TGase

50

Glycosylated hydrolysate 37 ˚C in the presence of TGase 0

Umami amino acid Fig. 4. (a) Comparison of the perceived saltiness (n = 64) and savouriness (n = 61) intensity of poultry protein hydrolysate in response to glucosamine treatments. The cross (+) represents the mean rank score in each box plot. Lower mean rank scores indicate higher perceived intensity and higher mean rank scores indicate lower perceived intensity. Means with different letters in the same sensory attribute are significantly different (p < 0.05). (b) Umami profile of poultry protein hydrolysate in relation to glucosamine treatment (n = 3). Total umami amino acids = free glutamic acid + free aspartic acid.

modifications to meat protein peptides. In Section 3.4, the discussion would focus on whether these modifications were sufficient to impart an appreciable effect on the perceived saltiness and savouriness or not. 3.4. Sensory evaluation and quantification of savoury free amino acids in relation to GlcN treatments In order to understand whether treating of hydrolysed poultry protein with GlcN had an effect on its sensory attributes, ranking tests were conducted. Specifically, the saltiness and the savouriness in seasoning compositions, acidified with acetic acid to a final pH of 4.9, were evaluated. It was hypothesised that production of

MRP through glycation with GlcN would enhance these two attributes in comparison to the untreated hydrolysate. Commercial taste enhancer formulations (seasoning controls), reported in Table 1, were also used as a comparison to the treatments. Fig. 4a presents the results from the first sensory panel, in which the saltiness of the seasoning samples was assessed. Despite several panellists commenting that the seasonings were too salty, the treatments subjected to GlcN glycation at 37 and 50 °C along with native hydrolysate incubated at 37 °C were ranked the highest intensity (p < 0.05) as compared to the negative control containing only NaCl. This implies a superior salt perception of the treatments that contain hydrolysed PPI. Moreover, the meat protein hydrolysate glycated at 37 °C without TGase was perceived

P.K. Hong et al. / Food Chemistry 197 (2016) 1143–1152

as more salty (p < 0.05) when compared to the seasonings formulated with the commercial taste enhancers. At the same time, glycation treatments were not different (p > 0.05) in terms of salty perception as compared to the native hydrolysate, indicating that the chemical modifications induced by the Maillard reaction at 37 and 50 °C were not robust enough to trigger the salt enhancement effects. In general, the majority of the samples which contained the hydrolysed meat proteins (except for the hydrolysate incubated at 50 °C) possessed salt-enhancing properties as compared to the seasoning formulated with addition of NaCl alone. This is likely due to the presence of free glutamic and aspartic acids along with other specific peptides, which have the ability to increase the salty sensation. The effectiveness of glutamic and aspartic acid to amplify saltiness is well-documented in the literature (Fuke & Ueda, 1996). The amount of free savoury amino acids (Glu + Asp) found in this study ranged from 93 to 139 mg/L (Fig. 4b) and was not statistically different among the glycation treatments and the native hydrolysates (p > 0.05). Nevertheless, the concentrations found in this work were low as compared to the ones reported in previous studies (Lioe, Takara, & Yasuda, 2006) in terms of triggering umami and salty sensation. However, the specific matrix composition formulated in this study which includes acetic acid at 13 mM may have played a role in amplifying the salty sensation. In addition, Maehashi et al. (1999) reported that chicken meat protein hydrolysate contains several acidic peptides with capacity to increase both the umami and salty sensation. In summary, the amounts of Glu and Asp in combination with acetic acid were able to generate a seasoning composition that increased the perceived saltiness compared to the seasoning containing only NaCl and acetic acid. Glycation, whether in the presence or absence of TGase did not increase the salty perception of the poultry meat protein hydrolysate. In the second sensory evaluation the savoury taste perceived in all of the seasoning compositions was not statistically different (p = 0.0593). These data were quite surprising as one would expect to find convergent results between salt intensity and savouriness, since the positive association between savouriness and saltiness has been proven before by Fuke and Ueda (1996). For instance, high savouriness is usually associated with high salt perception, a ‘‘formula” that has been applied to cut down the sodium level in processed food by subsequently increasing the amount of savoury compounds like, for instance, glutamic acid or IMP. In the current study, it appears that saltiness and savouriness are two independent attributes with minimal association. Panellists seemed to define saltiness differently from savouriness, except for one treatment, the hydrolysate glycated at 50 °C, where saltiness and savouriness were scored almost identically. This phenomenon could be ascribed to the fact that the untrained panellists rather than trained ones were recruited to evaluate the seasoning samples. Another possible reason could be due to the relative high level of sodium (Na+) used in the seasoning formulation (0.7% w/v). As a consequence, the addition of hydrolysed meat proteins to an already salty seasoning may have further increased the salty perception but not necessarily resulting in a balanced, savoury seasoning. For instance, the native hydrolysates which had a high salty perception, ranked at the bottom in terms of savouriness. In general, in spite of the analysis of variance for savoury perception which was not technically significant, the pvalue was in a range that could be considered a tendency (p = 0.0593). Among all of the treatments, glycation of meat hydrolysate conducted at 50 °C resulted in the greatest perceived savoury intensity, likely due to the greater production of a-DCs contributing to more MRP and thus the precursors of important flavour compounds (see Section 3.3). On the other hand, the native hydrolysates was the least savoury. In summary, despite the GlcN treatments being not as effective as expected in term of increasing

1151

saltiness and savouriness, the glycation conditions used in this study were rather mild in order to minimise production of AGEs, but also limiting the potential of GlcN to modify the meat protein hydrolysate. 4. Conclusions This study was able to demonstrate that poultry meat proteins hydrolysed with Alcalase had the capacity to enhance saltiness in seasoning compositions due a synergistic effect between the savoury amino acids (free Glu + free Asp) and acetic acid. Despite the GlcN-derived chemical modification of the hydrolysates, the enhancement on the perceived saltiness was not significant. However, a statistical tendency was observed in the perceived savouriness in response to GlcN treatment. A possible future improvement would be optimising the glycation conditions, such as increasing the concentration of GlcN and duration of the reaction. Conflict of interest There is no conflict of interest among the authors. Acknowledgements This work was supported by the Alberta Innovates Bio Solutions and the Alberta Livestock and Meat Agency Ltd. (Canada). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2015. 11.096. References Adler-Nissen, J. (1986). A review of food protein hydrolysis specific areas: Enzymic hydrolysis of food proteins. New York: Elsevier Applied Science Publications. Cheung, I. W. Y., & Li-Chan, E. C. Y. (2014). Application of taste sensing system for characterisation of enzymatic hydrolysates from shrimp processing byproducts. Food Chemistry, 145, 1076–1085. da Silva Ferreira, A. C., Reis, S., Rodrigues, C., Oliveira, C., & de Pinho, P. G. (2007). Silmultaneous determination of ketoacids and dicarbonyl compounds, key Maillard intermediates on the generation of aged wines aroma. Journal of Food Science, 72, S314–S318. Degen, J., Hellwig, M., & Henle, T. (2012). 1,2-Dicarbonyl compounds in commonly consumed foods. Journal of Agriculture and Food Chemistry, 60(28), 7071–7079. Eric, K., Raymond, L. V., Huang, M., Cheserek, M. J., Hayat, K., Savio, N. D., ... Zhang, X. (2013). Sensory attributes and antioxidant capacity of Maillard reaction products derived from xylose, cysteine and sunflower protein hydrolysate model system. Food Research International, 54, 1437–1447. Ferrer, E., Alegria, A., Farre, R., Clemente, G., & Calvo, C. (2005). Fluorescence, browning index, and color in infant formulas during storage. Journal of Agriculture and Food Chemistry, 53, 4911–4917. Fuke, S., & Ueda, Y. (1996). Interactions between umami and other flavor characteristics. Trends in Food Science and Technology, 7(12), 407–411. Hofmann, T., Bors, W., & Stettmaier, K. (1999). Studies on radical intermediates in the early stage of the nonenzymic browning reaction of carbohydrates and amino acids. Journal of Agriculture and Food Chemistry, 47(379–390). Hong, P. K., Gottardi, D., Ndagijimana, M., & Betti, M. (2014). Glycation and transglutaminase mediated glycosylation of fish gelatin peptides with glucosamine enhance bioactivity. Food Chemistry, 142(1), 258–293. Hrynets, Y., Ndagijimana, M., & Betti, M. (2013). Non-enzymatic glycation of natural actomyosin (NAM) with glucosamine in a liquid system at moderate temperatures. Food Chemistry, 139(1–4), 1062–1072. Hrynets, Y., Ndagijimana, M., & Betti, M. (2015a). Studies on the formation of Maillard and caramelization products from glucosamine incubated at 37 °C. Journal of Agriculture and Food Chemistry, 63(27), 6249–6261. Hrynets, Y., Ndagijimana, M., & Betti, M. (2015b). Rapid myoglobin aggregation through glucosamine-induced a-dicarbonyl formation. PLoS ONE, 10(9), e0139022. http://dx.doi.org/10.1371/journal.pone.0139022. Hrynets, Y., Omana, D. A., Xu, Y., & Betti, M. (2011). Comparative study on the effect of acid- and alkaline-aided extractions on mechanically separated turkey meat (MSTM): Chemical characteristics of recovered proteins. Process Biochemistry, 46(1), 335–343.

1152

P.K. Hong et al. / Food Chemistry 197 (2016) 1143–1152

Katsumata, T., Nakakuki, H., Tokunaga, C., Fujii, N., Egi, M., Phan, T. T., ... Lyall, V. (2008). Effect of Maillard reacted peptides on human salt taste and the amiloride-insensitive salt taste receptor (TRPV1t). Chemical Senses, 33, 665–680. Khiari, Z., Pietrasik, Z., Gaudette, N. J., & Betti, M. (2014). Poultry protein isolate prepared using an acid solubilisation/precipitation extraction influences the microstructure, the functionality and the consumer acceptability of a processed meat product. Food Structure, 2(1–2), 49–60. Kokkinidou, S. (2013). Inhibition of Maillard reaction pathways and off-flavor development in UHT milk: Structure reactivity of phenolic compounds. Duluth: University of Minnesota. Koo, S. Y., Bae, I. Y., Lee, S., Lee, D.-H., Hur, B.-S., & Lee, H. G. (2014). Evaluation of wheat gluten hydrolysates as taste-active compounds with antioxidant activity. Journal of Food Science and Technology, 51(3), 535–542. Lan, X., Liu, P., Xia, S., Jia, C., Mukunzi, D., Zhang, X., ... Xiao, Z. (2010). Temperature effect on the non-volatile compounds of Maillard reaction products derived from xylose–soybean peptide system: Further insights into thermal degradation and cross-linking. Food Chemistry, 120(4), 967–972. Lee, S. M., Jo, Y. J., & Kim, Y. S. (2010). Investigation of the aroma-active compounds formed in the Maillard reaction between glutathione and reducing sugars. Journal of Agriculture and Food Chemistry, 58, 3116–3124. Liao, L., Qiu, C., Liu, T., Zhao, M., Ren, J., & Zhao, H. (2010). Susceptibility of wheat gluten to enzymatic hydrolysis following deamidation with acetic acid and sensory characteristics of the resultant hydrolysates. Journal of Cereal Science, 52 (3), 395–403. Lioe, H. M., Takara, K., & Yasuda, M. (2006). Evaluation of peptide contribution to the intense umami taste of Japanese soy sauce. Journal of Food Science, 71(3), S277–S283. Maehashi, K., Matsuzaki, M., Yamamoto, Y., & Udaka, S. (1999). Isolation of peptides from an enzymatic hydrolysate of food proteins and characterization of their taste properties. Bioscience, Biotechnology, and Biochemistry, 63(3), 555–559. Matiacevich, S. B., & Buera, M. P. (2006). A critical evaluation of flourescence as a potential marker for the Maillard reaction. Food Chemistry, 95, 423–430. Morales, F. J., & Jimenez-Perez, S. (2001). Free radical scavenging capacity of Maillard reaction products as related to colour and fluorescence. Food Chemistry, 72, 119–125. Mottram, D. S., Wedzicha, B. L., & Dodson, T. A. (2002). Food chemistry: Acrylamide is formed in the Maillard reaction. Nature, 419, 448–449.

Ogasawara, M., Katsumata, T., & Egi, M. (2006). Taste properties of Maillard-reaction products prepared from 1000 to 5000 Da peptide. Food Chemistry, 99, 600–604. Papetti, A., Mascherpa, D., & Gazzani, G. (2014). Free a-dicarbonyl compounds in coffee, barley coffee and soy sauce and effects of in vitro digestion. Food Chemistry, 164, 259–265. Papetti, A., Mascherpa, D., Marrubini, G., & Gazzani, G. (2013). Effect of in vitro digestion on free a-dicarbonyl compounds in balsamic vinegars. Journal of Food Science, 78(4), C514–C519. Poulsen, M. W., Hedegaard, R. V., Andersen, J. M., de Courten, B., Bugel, S., Nielsen, J., ... Dragsted, L. O. (2013). Advanced glycation endproducts in food and their effects on health. Food Chemical Toxicology, 60, 10–37. Schlichtherle-Cerny, H., & Amado, R. (2002). Analysis of taste-active compounds in an enzymatic hydrolysate of deamidated wheat gluten. Journal of Agriculture and Food Chemistry, 50, 1515–1522. Sedgwick, S. W., Fenton, T. W., & Thompson, J. R. (1991). Effect of protein precipitating agents on the recovery of plasma free amino acids. Canadian Journal of Animal Science, 71, 953–957. Song, N., Tan, C., Huang, M., Liu, P., Eric, K., Zhang, X., ... Jia, C. (2013). Transglutaminase cross-linking effect on sensory characteristics and antioxidant activities of Maillard reaction products from soybean protein hydrolysates. Food Chemistry, 136, 144–151. Stadler, R. H., Blank, I., Varga, N., Robert, F., Hau, J., Guy, P. A., ... Riediker, S. (2002). Acrylamide from Maillard reaction products. Nature, 449–450. Thornalley, P. J., Langborg, A., & Minhas, H. S. (1999). Formation of glyoxal, methylglyoxal and 2-deoxyglucose in the glycation of proteins by glucose. Biochemical Journal, 344, 109–116. Weenen, H. (1998). Reactive intermediates and carbohydrate fragmentation in Maillard chemistry. Food Chemistry, 62, 393–401. Wilker, S. C., Chellan, P., Arnold, B. M., & Nagaraj, R. H. (2001). Chromatographic quantification of argpyrimidine, a methylglyoxal-derived product in tissue proteins: Comparison with pentosidine. Analytical Biochemistry, 290(2), 353–358. Xu, Q., Liu, J., Song, H., Zou, T., Liu, Y., & Zhang, S. (2013). Formation mechanism of volatile and non-volatile compounds in peptide-xylose Maillard reaction. Food Research International, 54(1), 683–690. Yaylayan, V. A., & Keyhani, A. (2000). Origin of carbohydrate degradation products in L-alanine/D-[13C]glucose model systems. Journal of Agriculture and Food Chemistry, 48, 2415–2419.